Abstract. The goal of our project is to develop noninvasive, safe, temporal monitoring of adeno- associated viral vector biodistribution following in vivo administration that can be ultimately used in humans. Our strategy is to covalently radioiodinate AAV capsids using the positron emitting isotope iodine-124 (I-124) and track the labeled capsid using positron emission tomography (PET). Our preliminary data in mice, rats and nonhuman primates has demonstrated the potential of assessing capsid biodistribution as a function of time for at least 10 days after administration. We introduce the concept of in vivo viral vector dosimetry, defined in the context of 3-dimensional spatial mapping of viral vector biodistribution at sub-centimeter spatial resolution anywhere in the body. The application of formal dosimetry principles will yield the number of capsids delivered to a target organ from one or multiple routes and administrations. The degree to which vector biodistribution is a surrogate for both on-target and off- target levels of transgene expression will be determined using biodistribution reporter gene standard assays against which we will compare the noninvasive imaging. We will evaluate 5 AAV serotypes, including 4 naturally occurring serotypes (AAV5, AAV8, AAV9, and AAVrh.10), that are commonly used in experimental animals and humans. As an example of a capsid modified AAV serotype engineered to alter vector biodistribution, we will study AAV5-pK2, an AAV5 capsid genetically modified with lysine residues, markedly altering vector biodistribution when administered intravenously. In aim 1 we will optimize our radio- labeling procedures and quality assurance procedures for the labeled vectors. In aim 2 we will assess each of several serotype I-124 labeled vectors in nonhuman primates with no pre-existing immunity to the serotype of the test vector, using the common intravenous and intracisternal delivery routes. In aim 3 we will investigate biodistribution in the context of immunity against each serotype. A direct benefit of the project will be the ability to noninvasively monitor the effectiveness of AAV vector administration relevant to a wide range of AAV gene therapy trials. Our methods should find special utility in rapidly assessing new capsid designs and different delivery routes for in vivo gene therapy. In addition, our methods will facilitate development of the new field of viral vector dosimetry, which will provide researchers and clinicians a quantitative tool for calculating vector delivery to organs in different gene therapy applications. Importantly, the methods that we will develop are designed to facilitate noninvasive imaging well-suited for translation to human use.